We present the adaptation of an IFCC method for α-amylase using 2-chloro-4-nitro-phenyl-α-D-maltotrioside as substrate (1) suited for routine work at 37℃. In the assay, a constant proportion of substrate, i. e. 92%, is directly converted to 2-chloro-4-nitrophenol and maltotriose. The method is based on multi- and univariate optimization leading to following measurement conditions: substrate, 2.25 mmol/l; chloride, 310 mmol/l; calcium 5.0 mmol/l; 4-morpholinoethanesulphonic acid, 50 mmol/l; pH 6.28. The assay may be carried out manually or by mechanized procedures, with substrate or sample start, and it shows these analytical properties in measuring amylase activity of sera: no lag phase, detection limit 2.9 U/l, linear range ≤820 U/l (for 300 s) or ≤1450 U/l (for 120 s of measurement), and total manual imprecision 3.2% (CV) at 46 U/l. Bilirubin ≤630 μmol/l, haemoglobin ≤6 g/l, triacylglycerols ≤30 mmol/l, heparin ≤100 kU/l, and glucose ≤120 mmol/l do not interfere. For adults, we established a preliminary 0.95-reference interval of 30–90 U/l not dependent on sex or age. A close association with the IFCC method demonstrates the reliable transfer of its measurement conditions to a robust routine method with minimal changes.